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Cloning the Vision Related G Protein Transducin for Live Cell Fluorescence Studies Deanna M

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Cloning the Vision Related G Protein Transducin for Live Cell Fluorescence Studies Deanna M The University of Akron IdeaExchange@UAkron Williams Honors College, Honors Research The Dr. Gary B. and Pamela S. Williams Honors Projects College Spring 2019 Cloning the vision related G protein transducin for live cell fluorescence studies Deanna M. Bowman The University of Akron, [email protected] Please take a moment to share how this work helps you through this survey. Your feedback will be important as we plan further development of our repository. Follow this and additional works at: https://ideaexchange.uakron.edu/honors_research_projects Part of the Biochemistry Commons, and the Other Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Bowman, Deanna M., "Cloning the vision related G protein transducin for live cell fluorescence studies" (2019). Williams Honors College, Honors Research Projects. 943. https://ideaexchange.uakron.edu/honors_research_projects/943 This Honors Research Project is brought to you for free and open access by The Dr. Gary B. and Pamela S. Williams Honors College at IdeaExchange@UAkron, the institutional repository of The nivU ersity of Akron in Akron, Ohio, USA. It has been accepted for inclusion in Williams Honors College, Honors Research Projects by an authorized administrator of IdeaExchange@UAkron. For more information, please contact [email protected], [email protected]. Title: Cloning the vision related G protein transducin for live cell fluorescence studies Name: Deanna Bowman Course: 3150:497 Date: April 26 2019 1 Abstract: G coupled protein receptors (GCPR) are one of the largest families of receptors and mediate a variety of biological responses. Rhodopsin is the largest family and aids in sight, the α-subunit of the GCPR complex in extremely important to the activation and downstream signaling effects of GCPR. The α-subunit contains a small trans-domain portion and in this project the sequence of that portion will be inserted into a vector containing a fluorescent tag. These vectors will then be used to collect fluorescent cross correlation spectroscopy or FCCS data. The unit was cloned using assembly methods that include PCR and purification techniques. The vector containing the transducin α-subunit is then transfected into live Cos-7 cells. Images of the cell can then be taken and FCCS data can also be collected. This will give insight to the dimerization and localization of the subunit in real time live cell experiments. GCPRs are extremely important cell receptors and insights on how molecular interactions between GPCRs and other proteins can lead to new routes for drug discovery. Abbreviations: GPCR: G protein-coupled receptor; Rho, rhodopsin; GDP, guanine diphosphate; GTP, guanine triphosphate; FCS, Fluorescence Correlation Spectroscopy; PIE FCCS, Pulsed-Interleaved Excitation Fluorescence Cross-Correlation Spectroscopy; GFP, green fluorescent protein; 2 Introduction: Guanine nucleotide binding protein (G protein) coupled receptors (GPCR) are not only one of the largest families of receptors found in biological systems but also mediate a large array of biological functions from sight to neurotransmitters.1 In this study, rhodopsin is examined, the rhodopsin family is the largest of the five GPCR families. The family shares a few conserved sequence motifs that are important for sight and mutations in rhodopsin have been found to contribute to night blindness.2 Within rod cells there are GPCRs in the discs that form the rod outer segment, with light activation in the transmembrane segment that contains a chromophore that undergoes photoisomerization.3 The light activation is by a single photon, causing the receptor, Rho, to undergo a conformational change as seen in Figure 1.4 The conformational change allows for 5 the light signal to be amplified by the heterotrimeric transducin molecule, Gt, and then dissociate. This conformational change is critical to the activation of the receptor and the subsequent signal transduction. Figure 1: This image shows GPCR on the cell membrane and the α-subunit. This image helps to visualize the activation of the Gα and how the signal transduction works. From Li, J. et al. (2002) Nature 420, 716-717. The alpha subunit (α-subunit, or Gα) is part of the heterotrimeric G proteins that associate with GCPRs when they are activated. The Gα subunit anchors to the plasma membrane and is important in the binding of GTP or GDP and specifically the amplification of the light signal in rhodopsin. The activation of the rod receptors is related to rhodopsin clustering, which could lead the receptors to localize to particular parts of the membrane effecting the kinetics of the receptor G protein interactions. In a recent study it was speculated that at high levels of illumination would reduce the availability of Gα to activate, this could lead to problems with eye sight.3 The Gα subunit is encoded by the gene GNAT1. This portion of the heterotrimeric G protein molecular interacts with the membrane in order to anchor the inactive Gα subunit that is bound to GDP. The molecule uses a N-myristoyl glycine on position 2 as a lipidation site to bind to the membrane.6 The heterotrimeric transducin domain also contains the binding site for GTP or GDP depending on the activation state of the receptor. As mentioned earlier, light absorption will activate conformational changes in rhodopsin that leads to the binding and activation of transducin to switch out GDP for GTP at the Gα subunit then dissociate from the rest of the 3 subunits. It has also been found that gene GNAT1 is highly conserved among different species and a missense mutation is often found in congenital stationary night blindness (CSNB).7 This mutation effects the activation of the receptor and the conformational changes needed to activate the signal. Figure 2 shows the structure for the Gα transducing domain complexed with GTP, this means that the protein is activated and able to dissociate from the membrane.8 Figure 2: In this figure the structure of the G alpha transducing domain is shown. This figure was created using the mmCIF file found on rcsb.org and software PyMOL. The code for the structure is 1TND. In this study Gα interaction with rhodopsin will be examined with the technique called fluorescence correlation spectroscopy (FCS) and pulse-interleaved excitation fluorescence cross correlation spectroscopy or PIE FCCS.9 PIE FCCS uses fluorescently labeled molecules to determine dimerization and activation of membrane bound receptors, this allows for quantification of rhodopsin interaction in situ. This method works by using time-correlated photon counting with fluorescent imaging using custom developed equipment. With PIE FCCS one can determine the protein concentration, mobility, and clustering.9 This clustering can be analyzed and the order can be determined to be either a monomer, dimer, trimer, or oligomer. This is an extremely powerful and accurate tool since the experiments can be done in live cells without the need to fix the cells. For FCS, a single fluorescent molecule is examined, while with PIE FCCS a two-color fluorescence is evaluated. The spatial organization of the rhodopsin receptor in a dimer or higher order oligomer could affect the transduction of the light signal. The analysis of the data takes into account the different combinations of the labeled proteins that are dimerizing or forming higher order complexes on the membrane. In this experiment I will create a clone of the transducing Gα domain that will be inserted into a vector containing a GFP tag that will allow for FCS and FCCS data to be taken on the dimerization and activation of rhodopsin. The vector will be created by means of two different methods, one is a subcloning method with an 4 insertion into the relevant expression vectors while the second method is purchasing a plasmid already containing the GNAT1 gene and a GFP tag. This will allow us to measure the interaction strength and kinetics of Gα and rho. Materials and Methods: Constructs: In order to insert the gene of interest into our florescent vectors, sub-cloning was done with the NEBuilder HiFi DNA Assembly kit. The gene of interest was purchased from AddGene, it was XE101 alpha transducing CS2+ (catalog number 16681). Primers were then designed so that half of the primer would overlap with the gene of interest (GNAT1) and the other half would overlap with the insertion vector. The forward primer being TGAACCGTCAGATCCATGGGGGCTGGGGC and the reverse primer being CGACTGCAGAATTCGTCAGAAGAGGCCACAGTCTTTGAGGTTCTCC with an annealing temperature at 72. The underlined segment of the primer is the base coding for the GNAT1 gene that was in the vector provided by AddGene. The primers where then ordered from GeneWiz. First our empty vectors, pAcGFP1-N1 and pAcmcherry-N1, were digested with Nhel (NEB) and HindIII (NEB) to linearize it for easier amplification during the PCR steps. After each digestion the plasmid was purified to remove any impurities. The vector bought from AddGene containing the gene of interest was also digested with Hind III and Nhel. After purification, the GNAT1 vector was then amplified with the overlapping primers using PCR. The PCR protocol was as follows: a hot start at 98ͦ C for 30 seconds, 98ͦ C for 10 seconds to denature, 72ͦ C for 20 seconds to allow for annealing, then one minute at 72ͦ C, these three steps are repeated 30 times. Then the mixture is held at 72ͦ C for two minutes and then the PCR product is held at 4ͦ C indefinitely. The product was then ran on a gel (agarose from Bio-Rad)to separate out the product containing the gene from linearized vectors or primer dimers. The NEBuilder HiFi DNA Assembly master mix is then added to a solution containing the PCR product and the pAcGFP1-N1 vector to anneal the DNA back together. This was then purified. The assembly product was then transformed to competent E. coli, DH5α, and plated and allowed to grow for twenty- four hours.
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